Microscope and Method for Forming a Microscopic Image with an Extended Depth of Field

Abstract

The present invention concerns a method for producing a microscopic image with an extended depth of field by means of a microscope. The microscope comprises an images sensor that comprises pixels that are arranged as a matrix that is formed by lines. In a step of the method, a plurality of microscopic frames of a specimen is acquired while a focus position (z) is changed. The microscopic frames are acquired line by line. The focus position (z) is changed over a course of acquiring individuals of the microscopic frames. In a further step, parts of individuals of the acquired lines are identified. These parts sharply image the specimen. The identified parts of the lines are composed in order to form a microscopic image of the specimen with an extended depth of field. Furthermore, the present invention concerns a microscope.

Claims

1. A method for forming a microscopic image with an extended depth of field by means of a microscope that comprises an images sensor, wherein the image sensor comprises pixels that are arranged as a matrix that is formed by lines, wherein the method comprises the following steps: acquiring a plurality of microscopic frames of a specimen while a focus position (z) is changed, wherein the microscopic frames are acquired line by line, and wherein the focus position (z) is changed over a course of acquiring individuals of the microscopic frames; identifying parts of individuals of the acquired lines that sharply image the specimen; and composing the identified parts of the lines in order to form a microscopic image of the specimen with an extended depth of field.

2. The method according to claim 1, wherein the plurality of microscopic frames is acquired by using a rolling shutter.

3. The method according to claim 1, wherein the focus position (z) is changed by changing a distance between the specimen and an objective of the microscope.

4. The method according to claim 1, wherein the focus position (z) is changed periodically, wherein a frequency of the periodical change is at least 100 Hz.

5. The method according to claim 4, wherein the plurality of microscopic frames is acquired during at least ten periods of the periodical change of the focus position.

6. The method according to claim 1, wherein the focus position (z) is changed stepwise.

7. The method according to claim 1, wherein the plurality of microscopic frames comprises at least 20 of the microscopic frames.

8. The method according to claim 1, wherein it further comprises the following step: recording a value of the focus position for each of the parts of the lines of each of the microscopic frames; wherein the composition of the identified parts of the lines in order to form a microscopic image of the specimen with an extended depth of field is based on the recorded values of the focus position (z) assigned to the identified parts of the lines.

9. The method according to claim 8, further comprising the following step: ascertaining values of height for the microscopic image with an extended depth of field based on the recorded values of the focus position (z) assigned to the identified parts of the lines.

10. The method according to claim 9, further comprising the following step: ascertaining a microscopic three-dimensional image of the specimen based on the microscopic image with an extended depth of field and based on the values of height.

11. The method according to claim 8, wherein the focus position (z) is controlled in order to change the focus position (z) based on a command value of the focus position, wherein the command value is used for recording a value of the focus position (z) for each part of the lines of each of the microscopic frames.

12. The method according claim 1, wherein the parts of the acquired lines of the microscopic frames are identified by searching maxima (max) of a signal of an image sensor over time.

13. The method according to claim 1, wherein the plurality of the microscopic frames is acquired at a frame rate of at least 10 frames per second.

14. The method according to claim 1, wherein it is repeated periodically in order to form a sequence of the images with an extended depth of field, wherein the images with an extended depth of field are formed at a rate of at least ten images per second.

15. A microscope, comprising: an objective lens for optically imaging a specimen; at least one actuator for changing a focus position (z) of the microscope; and a control and image processing unit being configured to carry out a method according to claim 1.

16. The microscope according to claim 15, wherein at least one actuator further comprises a microsystem for mechanically moving micromirrors and/or microlenses.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Additional advantages, details and refinements of the invention will become apparent from the following description of preferred embodiments of the invention, making reference to the drawing. There are shown:

[0051] FIG. 1 is a diagram of signal intensity over time representing a preferred embodiment of a method according to the invention;

[0052] FIG. 2 is a diagram of signal intensity over coordinate z representing a method according to prior art;

[0053] FIG. 3 is a diagram of acquiring microscopic frames according to a preferred embodiment of the method according to the invention; and

[0054] FIG. 4 is a diagram of acquiring microscopic according to prior art.

DETAILED DESCRIPTION OF THE INVENTION

[0055] FIG. 1 shows a diagram of signal intensity A over time t representing a preferred embodiment of a method according to the invention. The signal intensity A is measured at an output of an image sensor (not shown) of a microscope (not shown). The signal intensity A is related to a local position of a pixel of the image sensor. The signal intensity A depends on time t since a plurality of microscopic frames is acquired over time t. A focus position at a coordinate z of the microscope (not shown) is changed, e. g., periodically or randomly. Hence, time t and coordinate z are linked. The image sensor (not shown) operates according to a method of a rolling shutter. The image sensor (not shown) is preferably a CMOS sensor.

[0056] The signal intensity A shows a global maximum max at z=z.sub.1. At z.sub.1, the pixel of the corresponding frame acquired by the image sensor (not shown) is sharp. Due to the rolling shutter, there are also local maxima as, e. g., max.sub.loc. At these local maxima max.sub.loc, the pixel of corresponding frame acquired by the image sensor (not shown) is also sharp. The sharp pixels at the global maximum are composed to a microscopic image with an extended depth of field. Optionally, the sharp pixels at the local maxima max.sub.loc are composed to further microscopic images with an extended depth of field.

[0057] FIG. 2 shows a diagram of signal intensity A over coordinate z representing a method according to prior art. The signal intensity A is measured at an output of an image sensor (not shown) of a microscope (not shown). The signal intensity A is related to the local position of a pixel of the image sensor. The signal intensity A depends on time t since a plurality of microscopic frames is acquired over time t. A focus position at a coordinate z of the microscope (not shown) is changed periodically. The image sensor (not shown) operates according to a method of a global shutter. The image sensor (not shown) is, e. g., a CCD sensor. Due to the global shutter, the signal intensity A shows only one maximum max at z=z.sub.1. At z.sub.1, the pixel of corresponding frame acquired by the image sensor (not shown) is sharp.

[0058] FIG. 3 shows a diagram of acquiring microscopic frames according to a preferred embodiment of the method according to the invention. The four microscopic frames are numbered as N, N+n, N+m, and N+p. The four microscopic frames N. N+n, N+m, N+p are acquired over time t at t.sub.0, t.sub.n, t.sub.m, and t.sub.p. As explained above referring to FIG. 1, the focus position at the coordinate z is changed, e. g., randomly. Since the image sensor (not shown) operates according to a method of a rolling shutter, the lines and parts of the lines, respectively, of each individual microscopic frame N, N+n, N+m, N+p are acquired at different values z.sub.0, z.sub.1, z.sub.2, and z.sub.3 of the focus position at the coordinate z.

[0059] FIG. 4 shows a diagram of acquiring microscopic according to prior art. The four microscopic frames are numbered as N, N+1, N+2, and N+3. The four microscopic frames N, N+1, N+2, N+3 are acquired over time t at t.sub.0, t.sub.1, t.sub.2, and t.sub.3. As explained above referring to FIG. 2, the image sensor (not shown) operates according to a method of a global shutter. Due to the global shutter, every individual microscopic frame N, N+1, N+2, N+3 was acquired at a single value of the focus position at the coordinate z, namely, at z.sub.0, z.sub.1, z.sub.2, and z.sub.3, respectively.

[0060] In an exemplary embodiment of the method according to the invention, a stack of 20 to 60 microscopic frames of a specimen is acquired while the focus position is changed periodically at a frequency of approximately 100 Hz. The microscopic frames are acquired at a rate of 60 frames per second. Pixels of the acquired lines of the frames that sharply image the specimen are composed to a microscopic image with an extended depth of field.